Methods for selecting enzymes having enhanced activity

Abstract

Provided herein are methods and means for enhancing enzymatic activity. The system makes use of an emulsion for in vitro compartmentalization of a library of synthetic compounds which have a gene and a marked enzymatic substrate both directly linked to a solid phase. Expressed enzymes with greater activity will preferentially release the selectable marker from the solid phase, whereas enzymes with less activity will leave the markers intact. Removal of the marked compounds provides an enriched gene library encoding for more active variants. Also described are synthetic compounds and emulsions which can be used in the methods.

1 . A method of selecting for a polypeptide having enzyme activity, the method comprising:
 (i) suspending a plurality of synthetic compounds in an aqueous phase, wherein the synthetic compounds individually comprise:
 (a) a polynucleotide encoding for a polypeptide; 
 (b) a solid phase linked to said polynucleotide; 
 (c) an enzyme substrate linked to said solid phase; and 
 (d) a selectable marker linked to said substrate; 
    wherein the aqueous phase comprises components for expression of the polypeptide;   (ii) forming a water-in-oil emulsion with the aqueous phase, wherein the synthetic compounds are compartmentalized in aqueous droplets of the emulsion;   (iii) expressing the polypeptides within the aqueous droplets of the emulsion, wherein a polypeptide with enzymatic activity toward the substrate in an aqueous droplet releases the selectable marker(s) in that droplet; and   (iv) separating the synthetic compounds to recover synthetic compounds comprising the selectable marker and/or synthetic compounds wherein the selectable marker has been released.   
     
     
         2 . The method of  claim 1 , wherein no more than 20% of the aqueous droplets of the water-in-oil emulsion comprise more than one synthetic compound. 
     
     
         3 . The method of  claim 1 , wherein the emulsion comprises at least about 10 6  aqueous droplets/mL of emulsion. 
     
     
         4 . The method of  claim 1 , wherein the selectable marker is an affinity tag. 
     
     
         5 . The method of  claim 1 , wherein the synthetic compounds comprising the selectable marker of step (iv) are separated with streptavidin. 
     
     
         6 . The method of  claim 1 , wherein the solid phase is a microbead or particle. 
     
     
         7 . The method of  claim 6 , wherein the solid phase is a hydrophobic microbead. 
     
     
         8 . The method of  claim 1 , wherein the solid phase is a gold nanoparticle. 
     
     
         9 . The method of  claim 1 , further comprising amplifying one or more polynucleotides of the synthetic compounds wherein the selectable marker has been released of step (iv). 
     
     
         10 . The method of  claim 1 , further comprising cloning one or more polynucleotides of the separated synthetic compounds from step (iv) into an expression vector. 
     
     
         11 . The method of  claim 10  further comprising transforming said expression vector into a recombinant host cell. 
     
     
         12 . A synthetic compound comprising:
 (a) a polynucleotide encoding for a polypeptide;   (b) a solid phase linked to said polynucleotide;   (c) an enzyme substrate linked to said solid phase; and   (d) a selectable marker linked to said substrate.   
     
     
         13 . The synthetic compound of  claim 12 , wherein the selectable marker is an affinity tag. 
     
     
         14 . The synthetic compound of  claim 12 , wherein the solid phase is a microbead or particle. 
     
     
         15 . The synthetic compound of  claim 14 , wherein the solid phase is a hydrophobic microbead. 
     
     
         16 . The synthetic compound of  claim 12 , wherein the solid phase is a gold nanoparticle. 
     
     
         17 . A method of making the synthetic compound of  claim 12 , comprising:
 (i) linking the solid phase to the polynucleotide encoding for a polypeptide;   (ii) linking the enzyme substrate to the solid phase;   (iii) linking the selectable marker to the substrate; and   (iv) recovering the synthetic compound.   
     
     
         18 . A polynucleotide library comprising a plurality of different synthetic compounds according to  claim 12 . 
     
     
         19 . A water-in-oil emulsion comprising the polynucleotide library of  claim 18 , wherein the synthetic compounds are compartmentalized in aqueous droplets of the emulsion. 
     
     
         20 . A method of making the emulsion of  claim 19 , comprising:
 (i) suspending the plurality of synthetic compounds in the aqueous phase; and   (ii) mixing the suspension of (i) with an oil.